Comparison of droplet digital PCR and real-time quantitative PCR for lentiviral vector titration

We have developed recombinant simian immunodeficiency virus (rSIV) pseudotyped with Sendai virus F and HN proteins (rSIV.F/HN) for treatment of lung diseases.

To facilitate clinical advancement, we routinely produce highly purified (anion-exchange/TFF), highly concentrated (target >1e9 TU/mL) Lots of rSIV.F/HN carrying a range of transgenes. We typically use real-time quantitative PCR (qPCR) assays for titration of vector particles (VP/mL; via Reverse-Transcription (RT)-qPCR quantification of viral RNA extracted from vector Lots) as well as transducing units (TU/mL; via quantification of integrated vector genome copies on genomic DNA extracted from cells transduced with serial dilutions of vector), essential factors for quality control and experimental dosing.

In contrast to qPCR, droplet digital PCR (ddPCR) does not require a standard curve and is reported to be more precise and reproducible. We compared qPCR and ddPCR for quantification of VP/mL and TU/mL titres (and respective P:I ratio) of n=18 crude (non-purified/non-concentrated) and n=54 highly purified/highly concentrated vector Lots. Using ddPCR we observed significantly lower VP/mL (~1.6-fold; p<0.0001), TU/mL (~2-fold; p<0.0001) and P:I ratios (~2.5-fold; p<0.0001) for purified Lots. Similarly, when determined by ddPCR, TU/mL titres of crude vector Lots (but not VP/mL titres) were also significantly lower (~2-fold; p<0.001).

Our ddPCR assays (i) showed significantly lower variability (p=0.036 & p=0.0245 for purified and crude vector Lots, respectively) compared with qPCR; (ii) avoided assay failure due to poor standard curve quality, and (iii) allowed TU/mL determination for n=6 crude Lots not quantifiable by qPCR. These data highlight the advantages of ddPCR for quantification of recombinant lentivirus.